BASIC AND TRANSLATIONAL PANCREAS

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1 GASTROENTEROLOGY 2012;143: T Cells That Target Carcinoembryonic Antigen Eradicate Orthotopic Pancreatic Carcinomas Without Inducing Autoimmune Colitis in Mice MARKUS CHMIELEWSKI,* OLGA HAHN,* GUNTER RAPPL,* MICHAEL NOWAK, INGO H. SCHMIDT WOLF, ANDREAS A. HOMBACH,* and HINRICH ABKEN* *Center for Molecular Medicine Cologne, University of Cologne and Clinic I Internal Medicine, University Hospital Cologne, Cologne; and Department of Internal Medicine III, University Hospital Bonn, Bonn, Germany BACKGROUND & AIMS: New treatment approaches are needed for patients with pancreatic adenocarcinoma. Carcinoembryonic antigen (CEA) is highly expressed on the surface of pancreatic adenocarcinoma cells; we investigated the effects of cytolytic T cells that recognize CEA in a mouse model of pancreatic carcinoma. METHODS: Immune-competent mice that expressed the CEA transgene (CEAtg) in the intestinal and pulmonary tracts were given intrapancreatic injections of Panc02 CEA cells (express CEA and click beetle luciferase) and tumors were grown for 10 days. Mice were then given single intravenous injections of T cells engineered to express a chimeric antigen receptor (CAR) with high specificity, but moderate affinity, for CEA and a luminescence marker. RE- SULTS: Injection of the anti-cea CAR T cells reduced the size of pancreatic tumors to below the limit of detection in all mice and produced long-term tumor eradication in 67% of mice. T cells also eradicated CEA fibrosarcoma cells injected 45 days later. Bioluminescence imaging revealed the accumulation and persistence of the T cells at the tumor site. The efficacy of the T cells did not require lymphodepletion and was not reduced by soluble CEA. Mice developed some noninflammatory infiltrations of CAR T cells in intestine and lung, but there was no evidence of destruction of CEA healthy tissues. CON- CLUSIONS: Injection of T cells that target CEA can eradicate tumors grown from CEA pancreatic carcinoma cells in the pancreas of CEAtg mice without autoimmune effects. Keywords: Chimeric Antigen Receptor; Adoptive Cell Therapy; CEA; Immune Cell Therapy. Pancreatic cancer is the fourth most common cause of cancer death worldwide and is mostly detected at an advanced stage of the disease with no hope for primary curative therapy. 1 At diagnosis, patients with pancreatic cancer expect a mean survival time of less than 2 years, having the worst prognosis of any major malignancy. Based on the assumption that the cellular immune system is capable of controlling tumor development and progression in the long term, research aims at improving specific pancreatic cancer cell recognition and elimination by cytolytic immune cells, in particular, cytotoxic T cells. Strategies are currently being explored to adoptively transfer T cells engineered with a recombinant T-cell receptor (TCR) or a chimeric antigen receptor (CAR) recognizing a defined tumor-associated antigen, aiming at redirecting the patient s T-cell response specifically toward the malignant lesion. Such modified T cells showed remarkable efficacy in recent trials. 2,3 In contrast to the TCR, the CAR consists of one polypeptide chain with an antibody-derived extracellular domain for target binding and a TCR-derived intracellular signaling domain for T-cell activation upon target engagement. 4 Due to the antibody-derived targeting domain, CAR-modified T cells recognize their target independent of major histocompatibility complex presentation and can potentially target any antigen for which an antibody is available. 4 Carcinoembryonic antigen (CEA) seems to be a valuable target for the specific immunotherapy of pancreas carcinoma by adoptive therapy with CAR-modified T cells because CEA is highly expressed on the surface of a majority of pancreatic adenocarcinomas. 5 CEA expression is elevated in nearly 90% of cancers arising from endodermal tissue, including the gastrointestinal tract, pulmonary tissues, and breast. 6 A series of CARs targeting CEA were reported during the last several years, each targeting different CEA epitopes. For example, the MFZ and the H10 CAR target the membrane distal N domain, whereas the BW431/26 scfv CAR targets the membrane-proximal A3 domain of the CEA molecule. 7 Like most tumorassociated antigens, however, CEA is not tumor selective and is physiologically expressed on healthy epithelia of the gastrointestinal tract and the lung. After neoplastic transformation, luminal epithelia cells lose the apical polarity of CEA expression with the consequence that CEA gains access to blood capillaries, becomes visible to im- Abbreviations used in this paper: CAR, chimeric antigen receptor; CBLuc, click beetle luciferase; CEA, carcinoembryonic antigen; GLuc, Gaussia luciferase; IL, interleukin; mab, monoclonal antibody; MPO, myeloperoxidase; TCR, T-cell receptor; tg, transgenic; TGF-, transforming growth factor by the AGA Institute /$

2 1096 CHMIELEWSKI ET AL GASTROENTEROLOGY Vol. 143, No. 4 mune cells, and produces increased serum levels of soluble CEA, the latter is used to monitor disease progression in patients with CEA carcinoma. Targeting CEA by CARredirected T cells has the potential to damage healthy tissues, resulting in severe autoimmunity. A recent trial aimed at eliminating CEA metastatic colorectal cancer by adoptive therapy with anti-cea CAR T cells induced severe autoimmune colitis and pneumonia in all 3 patients, which, although transient, represented dose-limiting toxicity and required withdrawal from therapy. 2 These and other clinical observations point out the challenge to target CEA pancreatic carcinoma without inducing a therapy-limiting autoimmune response. Based on a clinically relevant model, we report on successful treatment of orthotopic CEA pancreatic adenocarcinoma by adoptive transfer of anti-cea CAR-engineered T cells. We proofed T-cell therapy for safety in the CEA transgenic (tg) mouse, which closely mimics the human situation with respect to physiological CEA expression in the gastrointestinal and pulmonary tract, soluble CEA in serum, and immunological tolerance toward CEA. 8 This is the first report to demonstrate in a clinically relevant model that anti-cea T cells, when redirected by the SCA431-antibody driven CAR, can specifically and efficiently eliminate CEA pancreas carcinoma without inducing substantial autoimmunity. The observation is of particular relevance with respect to both a clinically feasible strategy to control so far untreatable CEA pancreas carcinoma and to avoid therapy-limiting autoimmunity. Methods Cell Lines 293T cells are human embryonic kidney cells that express the SV40 large T antigen (ATCC CRL-11268). Panc02 is a tumorigenic murine pancreatic carcinoma line. 9 The Panc02 CEA cells were derived from Panc02 cells by transfection with plasmid-encoding human CEA (kindly provided by Dr David Gilham, University of Manchester, UK). The Panc02 CEA and the Panc02 CEA cells coexpress green fluorescence protein and click beetle luciferase (CBLuc). MC38 is a murine fibrosarcoma line; C15A3 is a CEA derivative obtained after DNA transfection. MD45 is an established murine T-cell line. Pancreas Carcinoma Model CEAtg C57BL/6 mice were obtained from the Patterson Institute, Manchester, UK. 8 Murine pancreatic tumors with CEA and CBLuc expression were induced in 10-week-old CEAtg mice by intrapancreatic injection of Panc02 CEA cells. For in vivo bioluminescence imaging, CEA Panc02 cells were genetically modified with the CBLuc and cotransfected with the hygromycin B phosphotransferase gene for clonal selection. For in vivo T-cell imaging, CAR-modified T cells were retrovirally transduced with Gaussia luciferase (GLuc), which is anchored to the T-cell membrane by the CD8 transmembrane domain. In vivo imaging was performed upon intraperitoneal injection of D- luciferin (1.5 mg/mouse) or benzyl-coelenterazine (100 g/ mouse) (PJK GmbH, Kleinblittersdorf, Germany) as substrate for the CBLuc and GLuc, respectively, and visualized using the Photon Imager (Biospace Lab, Paris, France). The threshold of bioluminescence signals was automatically determined using Photo Vision software (Biospace Lab). Bioluminescence signals were accordingly filtered against background noise. Regions of interest were defined as regions above threshold and automatically gated by predefined program tools. There was no manual gating of regions of interest in order to avoid any incoherence. Photon emission intensity (photon/s/sr) was calculated from data of emitted photons from the respective regions of interest using the Photo Vision software. Magnetic-Activated Cell Sorting T cells were isolated from peripheral blood by magneticactivated cell sorting using mouse CD3 or CD8 Micro Beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Retroviral Modifications CARtg T cells from mouse spleen were preactivated with anti mouse CD3e mab, clone 145 2C11 (200 ng/ml), and antimouse CD28 mab, clone (100 ng/ml) (BD Biosciences, Mountain View, CA), respectively. T cells were cultivated in RPMI 1640 medium (Dutch modification) (Invitrogen GmbH, Darmstadt, Germany), 10% (v/v) fetal calf serum (Life Technologies, Paisley, UK), mouse IL-15 (5 ng/ml; Biolegend, San Diego, CA), 1% (v/v) nonessential amino acids and 2% (v/v) L-glutamate (both from Invitrogen). For generation of retroviral particles, the packaging cell line 293T was cotransfected with the Moloney murine leukemia virus based helper plasmids pvpack-eco and pvpack-gp coding for the retroviral proteins gag, pol, env (Stratagene, La Jolla, CA) and with the retroviral expression plasmid pbullet-gluc-cd8tm. Anti-CEA CAR-modified T cells were obtained from the spleen of the CARtg mouse. The SCA431scFvmIgG-CD28-CD3 CAR is composed of the CEA binding domain SCA431scFv linked by a murine IgG-domain to the murine intracellular CD28-CD3 signaling domain (Figure 1A). For GLuc modification, CARtg T cells were transduced with the respective retrovirus for 48 hours. Cytokines The Luminex-based Beadlyte mouse 6-Plex Multi-Cytokine Detection kit (Merck Millipore, Billerica, MA, USA) was used to measure cytokines in mouse sera and in supernatants from coculture experiments, respectively. Transforming growth factor (TGF- 1) and myeloperoxidase (MPO) were determined using the Max TGF- ELISA kit (BioLegend) and MPO kit (USCN Life Science, Wuhan, China), respectively, according to manufacturers instructions. CAR-Mediated T-Cell Activation To monitor the cytolytic activity, CAR-engineered CD8 T cells ( cells per well) were coincubated with tumor cells ( cells/well) for 36 hours in 96-well round-bottom plates. Specific cytotoxicity was monitored by XTT-based colorimetric assay (Roche Diagnostics, Mannheim, Germany). Immunohistological Analyses For CEA detection, cryostat sections were stained with the biotin-conjugated anti-cea mab CB30 (Ancell, Bayport, MN) and with streptavidin-horseradish peroxidase (BioLegend) and recorded with DAB chromogen substrate (Biozol, Eching, Germany). Sections were additionally stained with H&E (Carl Roth, Karsruhe, Germany). CAR-engineered T cells were detected

3 October 2012 ADOPTIVE THERAPY WITH CEA-REDIRECTED T CELLS 1097 Figure 1. T cells with CEA-specific CAR recognize and kill CEA Panc02 pancreas carcinoma cells. (A) Schematic diagrams depicting the modular composition of the CEA-specific CAR and the GLuc construct used for bioluminescence T-cell imaging. (B) Both CD4 and CD8 T cells were modified with the CAR and used for adoptive transfer in this study. CAR expression was monitored by flow cytometry using a phycoerythrin-labeled anti-mouse IgG1 antibody and an allophycocyanin-conjugated antibody directed against CD4 and CD8, respectively. CAR T cells expressed the CD62L (L-selectin), CD127 (IL-7R ) central memory phenotype and lack CD279 (PD-1), a marker for exhausted cells. (C) T cells with anti-cea CAR ( cells/well) were coincubated at the indicated effector-to-tumor cell ratios with Panc02 cells with or without CEA expression for 48 hours. Interferon-gamma in the culture supernatant as marker for T-cell activation was determined by enzyme-linked immunosorbent assay (ELISA). Cytotoxicity was assessed by an XTT-based viability assay. All measurements were performed in triplicate. (D) Supernatants from cocultures of T cells with Panc02 tumor cells were recorded for the inflammatory cytokines tumor necrosis factor, IL-17, IL-10, IL-5, IL-1, and interferon-gamma using the mouse multicytokine Milliplex assay as described in Methods. Secreted MPO and TGF- were recorded by ELISA, respectively. Displayed values represent the cytokine concentrations in supernatants of 10 5 T cells coincubated with 10 4 Panc02 cells. (E) Lung epithelia cells were isolated from the lung of CEAtg mice. Cells were stained for CEA and flow sorted before use in the study. Mouse CEA Panc02 pancreatic carcinoma cells and fibrosarcoma CEA C15A3 cells were used for comparison. (F) T cells with anti-cea CAR (10 5 cells) were cocultured with sorted CEA-positive and CEA-negative lung epithelia cells, and CEA Panc02 cells (10 4 cells), respectively, for 36 hours. Interferon-gamma in the supernatants was determined by ELISA. Data represent the mean of triplicate.

4 1098 CHMIELEWSKI ET AL GASTROENTEROLOGY Vol. 143, No. 4 Figure 2. The CEAtg mouse expressed CEA in transplanted CEA Panc02 pancreatic carcinoma as well as in healthy tissues. (A) Large and small intestine from CEAtg and wild-type (wt) C57Bl6 mice were stained with H&E and recorded for CEA expression. (B) Organs from the CEAtg mouse were stained for CEA and scanned by a whole slide scanner. (C) Soluble CEA (scea) in the blood serum of CEAtg and wt mice was determined by enzyme-linked immunosorbent assay. (D) CEA Panc02 pancreatic carcinoma cells ( cells/mouse) were inoculated into the pancreas of CEAtg mice. After 3 weeks, the tumor tissue, as well as the healthy pancreas (without tumor), were recorded by H&E and CEA staining.

5 October 2012 ADOPTIVE THERAPY WITH CEA-REDIRECTED T CELLS 1099 Figure 3. by the Dyelight 549-coupled, polyclonal goat anti-mouse -chain antibody (KPL Inc, Gaithersburg, MD). Central memory CD62L CD127 and exhausted CD279 T cells were recorded by staining cryostat sections with the Alexa-Fluor 488 conjugated anti-mouse CD62L mab MEL-14, with the Alexa-Fluor 647 anti-mouse CD127 mab A7R34 and with the Brilliant Violet 421 conjugated anti-mouse CD279 mab 29F.1A12 (all from BioLegend). Cryostat sections were stained with the Brilliant Violet 421 labeled anti-mouse Tumor necrosis factor mab MP6-XT22 (BioLegend). MPO was detected by staining with the rabbit anti-mouse MPO antibody (Abcam, Cambridge, UK) and the Cy5-conjugated anti-rabbit IgG (Abcam) antibody. The biotin-conjugated anti-mouse IL-1 mab B122 and Streptavidin Dyelight 488 (BioLegend) were used for IL-1 detection.

6 1100 CHMIELEWSKI ET AL GASTROENTEROLOGY Vol. 143, No. 4 Figure 3. (Cont d)

7 October 2012 ADOPTIVE THERAPY WITH CEA-REDIRECTED T CELLS 1101 Antibody Clonotype Screen Clonotype analysis was done using the SBA Clonotyping System-B6/C57J-HRP kit from Southern Biotechnology (Birmingham, AL), according to manufacturer s specifications. CEAspecific IgG 1 antibodies in the mouse sera were determined by enzyme-linked immunosorbent assay using the human recombinant CEA (Abcam) and the polyclonal horseradish peroxidase conjugated goat anti-mouse IgG 1 antibody (Southern Biotechnology). Results CAR-Engineered T-Cells Target CEA Pancreas Carcinoma Cells Mouse T cells were genetically engineered with the CEA-specific CAR that is entirely composed of murine domains and harbors the murine anti-cea scfv Sca421 in the extracellular domain for targeting, and the CD28 and CD3 signaling endodomains for T-cell activation (Figure 1A). Both CD4 and CD8 T cells expressed the CAR on the cell surface (Figure 1B); both modified T-cell subsets were present in the therapeutic T-cell inoculums in same frequencies as in the peripheral blood of healthy mice. CAR T cells predominantly expressed CD62L (L-selectin) and CD127 (IL-7R -chain) and lacked CD279 (PD-1) indicating central memory cells. To proof for CAR-mediated activation, modified T cells were coincubated in vitro with Panc02 mouse pancreas carcinoma cells, which lack CEA expression, and with CEA Panc02-derivative cells with engineered CEA expression. Anti-CEA CAR T cells lyse CEA Panc02 cells in a dose-dependent fashion, whereas CEA Panc02 cells were not eliminated (Figure 1C). Nonmodified T cells without CAR did not eliminate CEA or CEA Panc02 cells. CAR-mediated T-cell activation was accompanied by an increase in interferon-gamma secretion upon coincubation with CEA Panc02 cells, but not with CEA cells (Figure 1C). Adoptive Therapy With Anti-CEA CAR T Cells Eradicated Transplanted CEA Pancreas Carcinoma To evaluate the anti-tumor activity of a CEA-specific cell therapy in a clinically relevant situation, we used the immune-competent, CEAtg mouse, 8 which constitutively expressed CEA in the gastrointestinal and pulmonary tract (Figure 2A, B) and is tolerant for CEA. The CEAtg mouse also secreted soluble CEA into the serum, closely mimicking the human situation (Figure 2C). Panc02 pancreatic carcinoma cells were transplanted into the pancreas, giving rise to progressively growing adenocarcinomas. CEA Panc02 derivative cells likewise induced tumors of the same histology, however, with largely homogenous CEA expression (Figure 2D). Both carcinomas with and without CEA expression induced the death of mice within 3 weeks. When tumors were fairly established, ie, at day 10 after transplantation, autologous T cells with and without engineered anti-cea CAR were once adoptively transferred by intravenous injection into the tail vein. T cells with a CAR of irrelevant specificity and T cells without CAR served as controls. Mice did not receive lymphodepletion or other preconditioning before adoptive T-cell therapy. For in vivo imaging, T cells were marked with a membrane-anchored variant of GLuc and pancreatic carcinoma cells with CBLuc. After a single T-cell application, the tumor luciferase signal dropped under detectable levels, indicating substantial regression of pancreas carcinomas (Figure 3A). Tumor regression was exemplarily verified by autopsy and immune histological staining. Antitumor activity was efficient because tumor regression was observed in 8 mice and lasted for 20 weeks after T-cell treatment. The anti-tumor activity was specifically mediated by the CAR because CEA Panc02 tumors progressed upon anti-cea CAR T-cell application and T cells without CAR did not impact CEA Panc02 tumor progression (Figure 3A). In vitro incubation with the specific substrate 4 Fig. 3. Regression of transplanted pancreas carcinoma upon adoptive therapy with anti-cea CAR T cells. Pancreas carcinoma was initiated in the CEAtg mouse by intrapancreatic injection of CEA or CEA Panc02 carcinoma cells ( cells/mouse), both cells marked with CBLuc for bioluminescence imaging. When tumors were established, anti-cea CAR T cells marked with GLuc were injected into the tail vein at day 0 (10 7 cells/mouse). For comparison, T cells without CAR were injected. Up to 8 mice in each cohort were treated. (A) CBLuc-marked Panc02 tumor cells and GLuc-marked T cells were recorded by bioluminescence imaging in the same mouse at the indicated days before and after T-cell transfer. Data of bioluminescence signals were quantified as described in Methods; data represent the mean. Significance between the cohorts was analyzed by 2-tailed, unpaired Student t test ( * P.05; ** P.01). Freedom from tumors was determined for each cohort based on bioluminescence data for tumor detection. The significance between the cohorts was analyzed by 2-tailed, unpaired Student t test. (B) T cells marked with GLuc for use in bioluminescence imaging were recorded in vitro in presence of the GLuc substrate benzyl-coelenterazine ( cells/well) and recorded by a Photon Imager device. Unmodified cells served as control. (C) CEAtg mice with transplanted pancreatic tumors were treated with CAR GLuc T cells; T cells without CAR served as control. T-cell infiltration in the various tissues was recorded by bioluminescence 4 weeks after adoptive therapy. Gastrointestinal tissues and lung showed accumulation with CAR T cells, but not of T cells without CAR. (D) Tissues from CEAtg mice treated with CAR-engineered or nonmodified T cells were stained with H&E and recorded for CEA by antibody staining. Wild-type (wt) mice treated with CAR T cells were analyzed for comparison. (E) Organs from CEAtg mice treated with anti-cea CAR T cells were H&E stained, recorded for CEA, and scanned by use of a whole slide scanner. Slides from gastrointestinal tissues and the lung are exemplarily shown. No major pathology was recorded. (F) CAR T cells infiltrated the large and small intestine, the lung and the site of pancreatic tumor cell inoculation of CEAtg mice at day 23 after adoptive transfer as revealed by multicolor immune histology staining. CAR T cells in the lung and large and small intestine were predominantly of CD62L and CD127 central memory phenotype, whereas CAR T cells at the site of tumor cell inoculation mostly exhibited the CD279 -exhausted T-cell phenotype.

8 1102 CHMIELEWSKI ET AL GASTROENTEROLOGY Vol. 143, No. 4 Table 1. Histological Activity Index for Inflammation After Adoptive Cell Therapy CAR T cells in CEAtg mouse n T cells without CAR in CEAtg mouse n CAR T cells in WT mouse Stomach Small intestine Large intestine Lung Liver Spleen Organs from CEAtg mice treated with CAR-engineered (n 6) or nonmodified T cells (n 5) were stained with H&E and recorded for inflammatory infiltrates. Sections were evaluated by an independent expert taking into account the following criteria: Inflammatory activity Score Histopathology defining characteristics Inactive/quiescent/normal 0 No epithelial infiltration by neutrophils Mildly active 1 Neutrophil infiltration of 50% of sampled crypts or cross sections, no ulcers or erosions Moderately active 2 Neutrophil infiltration of 50% of sampled crypts or cross sections, no ulcers or erosions Severely active 3 Erosion or ulceration, irrespective of other features n validated that CAR T cells engineered with the membrane variant of GLuc produced a luciferase signal (Figure 3A). Taken together, adoptive cell therapy with anti-cea CAR T cells produces regression of an orthotopically transplanted, CEA pancreas carcinoma in the immune-competent mouse. No Induction of Autoimmune Pathology by Adoptive T-Cell Therapy With Anti-CEA CAR Anti-CEA CAR-modified T cells accumulated in the CEA pancreas carcinoma shortly after tail vein injection, as monitored by GLuc bioluminescence imaging, and persisted for at least 3 weeks (Figure 3A). T-cell accumulation was verified by bioluminescence of an isolated pancreas carcinoma and compared with T-cell infiltrations of the spleen or other organs (Figure 3C). Beside the gastrointestinal tract, anti-cea CAR T cells heavily infiltrated the lung. As control, T cells without CAR did not accumulate in the pancreas carcinoma, gastrointestinal tract, and in the lung. For comparison, anti-cea CAR T cells did not infiltrate the CEA-negative Panc02 tumor, but accumulated in the spleen (cf Figure 3A). Because CEA is expressed by luminal epithelial cells in the intestinal and pulmonary tracts of the CEAtg mouse, which mimics the human situation, we addressed whether adoptive cell therapy with anti-cea CAR T cells caused an autoimmune inflammatory response. Upon adoptive therapy with CAR-modified T cells, mice did not suffer from major alterations in their body weight; the same we observed after transfer of nonmodified T cells (Supplementary Figure 1). Mice with adoptively transferred CAR T cells and cured tumors had the same life expectancy as mice without tumor. The colon was not enlarged as expected for severe colitis. Histology screening of various organs, including stomach, liver, spleen, and lung, did not reveal major inflammatory cell infiltrations or tissue destruction (Figure 3D). Whole gut histology screening confirmed that anti-cea CAR T-cell therapy did not induce inflammatory destruction of the small and large intestine (Figure 3E). No epithelial infiltration by neutrophils in stomach, large and small intestine, and lung was observed (Histology Activity Index 0; Table 1). We conclude that eradication of CEA pancreatic carcinoma by adoptive cell therapy with anti-cea CAR T cells occurred without inducing autoimmune pathology (Supplementary Figure 1). CAR T cells used for adoptive cell therapy expressed a central memory phenotype indicated by CD62L (L-selectin) and CD127 (IL-7R -chain) expression and lack of CD279 (PD-1) (cf Figure 1B). Multicolor immune histology screening of various organs 23 days after therapy revealed infiltrations of CAR T cells with a central memory phenotype in the small and large intestine and in the lung (Figure 3F). Only a few CAR T cells expressed the exhausted PD-1 phenotype. In contrast, CAR T cells infiltrating the pancreatic tumor site predominantly expressed the CD62L CD127 PD1 phenotype of exhausted T cells, a minority were the central memory phenotype. CAR T cells at the tumor site were activated indicated by IL-1 and tumor necrosis factor expression, whereas intestine-infiltrating cells mostly lacked cytokine expression. Data imply that systemically applied central memory CAR T cells convert to exhausted cells at the tumor site, while preserving their phenotype in healthy organs with physiological CEA expression. Lymphodepletion Before T-Cell Therapy Did Not Favor Autoimmune Pathology Nonmyeloablative lymphodepletion before T-cell therapy improves T-cell expansion and is broadly used in adoptive cell therapy, including recent trials targeting CEA. 2 We therefore preconditioned tumor-bearing CEAtg mice by pretreatment with fludarabine and cycloheximide, resulting in the eradication of the lymphocyte compartment. Adoptive transfer of an anti-tumor dose of anti-cea CAR T cells, however, did not induce weight loss compared with mice treated with nonmodified T cells (Figure 4A). Those mice did not develop clinically detectable colitis during an observation time of 6 weeks. Immune histochemistry of the gut, however, revealed some T-cell infiltrations in the subepithe-

9 October 2012 ADOPTIVE THERAPY WITH CEA-REDIRECTED T CELLS 1103 therapy with anti-cea CAR T cells can produce some T-cell infiltrations in CEA tissues, however, without inducing clinically apparent autoimmune pathology. Soluble CEA Did Not Block Anti-CEA CAR-Redirected T-Cell Activation CEA is present in the serum of healthy individuals and in increased levels in CEA cancer patients to levels up to 20 g/ml. We therefore asked whether soluble CEA blocks the anti-cea CAR-mediated T-cell attack toward CEA tumor cells. Addition of CEA in a concentration of 25 g/ml or of bovine submaxillar mucin as control to anti-cea CAR T cells did not alter activation toward CEA cells, as indicated by the induction of IL-2 secretion (Figure 4C). To demonstrate specificity of redirected activation, T cells were blocked by the anti-idiotypic antibody BW2064, directed toward the anti-cea scfv domain, but not by an isotype-matched control antibody of irrelevant specificity. CEA used in the assay can specifically activate anti-cea CAR T cells because CEA immobilized on plastic surface induced IL-2 secretion of anti-cea CAR T cells, but not of nonmodified T cells (Figure 4D). Immobilized bovine submaxillar mucin as control did not activate anti-cea CAR T cells. We conclude that the CEA-redirected T cells are not blocked by soluble CEA, at least in concentrations commonly found in sera of cancer patients. Figure 4. Lymphodepletion before therapy did not favor autoimmune colitis upon CAR T-cell infusion. CEAtg mice were treated with cyclophospamide (200 mg per kg body weight) and fludarabine (150 mg per kg body weight) at day 3 and 2 before T-cell infusion to induce a nonmyeloablative lymphodepletion. (A) Body weight of mice did not substantially alter during treatment. (B) CEA and H&E staining of the large intestine from mice treated with CAR T cells or unmodified T cells did not reveal major histological pathologies. Soluble CEA did not block CEA-specific, CAR-redirected T-cell activation. (C) MD45 T cells ( cells) were engineered with anti-cea CAR and incubated for 48 hours with CEA Lovo colorectal carcinoma cells ( cells) in the presence of added soluble CEA (scea) or with soluble bovine submaxillary mucin (each 25 g/ml) or without additives (w/o) as controls. For comparison, the anti-cea scfv anti-idiotypic mab BW2046, which is directed against the CEA binding domain of the anti-cea CAR, and for control an anti-idiotypic mab of irrelevant specificity, were added (each 25 g/ml). (D) To confirm that CEA used in the assay can bind to the anti-cea CAR, MD45 T cells ( ) with or without (w/o) anti-cea CAR were incubated for 48 hours in microtiter plates coated with CEA or for control with bovine submaxillar mucin. IL-2 secreted into the culture supernatants indicating T-cell activation was recorded by enzyme-linked immunosorbent assay. lial colon wall of CAR T-cell treated mice, but not of mice treated with unmodified T cells (Figure 4B), implying that the anti-cea CAR T cells were redirected to the colon by their CAR. Taken together, we conclude that adoptive cell Mice That Successfully Rejected CEA Pancreatic Carcinoma Upon Adoptive T-Cell Therapy Rejected CEA Fibrosarcoma Cells Upon Secondary Challenge We explored secondary tumor cell rejection in mice that successfully eliminated transplanted CEA pancreatic carcinoma after adoptive therapy with anti-cea CAR T cells. Mice were challenged with both CEA C15A3 and CEA MC38 fibrosarcoma cells by subcutaneous injections in the right and left flanks, respectively, at day 45 after primary carcinoma cell inoculation. Fibrosarcoma cells were marked with CBLuc; C15A3 is the CEA derivative of the CEA MC38 fibrosarcoma cells. Bioluminescence imaging revealed rejection of CEA, but not of CEA, fibrosarcoma cells in all treated mice (Figure 5A), indicating a potent and CEA-specific secondary tumor cell rejection. As control, both tumor cell subsets produce tumors upon primary inoculation in mice lacking adoptive T-cell therapy. Secondary tumor cell rejection was accompanied by increases in serum levels of IL-1 and IL-5, whereas tumor necrosis factor, interferon-gamma, and TGF- 1 levels remained unaltered compared with those after the primary anti-tumor response (Figure 5B), indicating a T helper 1 dominated T-cell response. Serum levels of IL-17 and IL-10 also increased and MPO decreased. Secondary tumor cell rejection was accompanied by an increase in IgG1 antibody levels, whereas the amounts of the other immunoglobulin clonotypes, including IgG3, IgG2b, IgG2c, IgA, IgM or or light

10 1104 CHMIELEWSKI ET AL GASTROENTEROLOGY Vol. 143, No. 4 Figure 5. Mice that successfully eliminated CEA Panc02 pancreatic carcinoma by anti- CEA CAR T-cell therapy rejected CEA C15A3 fibrosarcoma cells upon short-term challenge. (A) CEAtg mice with CEA Panc02 tumors were treated with anti- CEA CAR T cells as described in Figure 3. Mice that successfully eliminated pancreatic tumors were subcutaneously injected at day 45 with 10 6 CEA CBLuc C15A3 at the right flank and CEA CBLuc MC38 fibrosarcoma cells at the left flank (6 mice). C15A3 is the CEA derivative of CEA MC38 cells. Tumor growth was recorded by CBLuc bioluminescence; freedom from tumor cell load was determined on the basis of bioluminescence data. The significance of data between left and right flank tumors was analyzed by 2-tailed, unpaired Student t test. (B) Blood sera were assayed for a panel of cytokines 5 days after pancreatic tumor cell inoculation, CAR T-cell treatment, and tumor cell challenge, respectively. The serum concentrations of tumor necrosis, IL- 17, IL-10, IL-5, IL-1, and interferon-gamma were determined using the mouse Milliplex assay. Secreted MPO and TGF- were measured by enzyme-linked immunosorbent assay (ELISA). (C) Sera from treated mice were analyzed for changes in antibody clonotypes by ELISA. (D) Serum CEA levels and CEA-specific IgG 1 antibodies in treated CEAtg mice were recorded 5 days after pancreatic tumor cell inoculation, CAR T-cell treatment and tumor-cell challenge. Mocktreated mice were analyzed for comparison.

11 October 2012 ADOPTIVE THERAPY WITH CEA-REDIRECTED T CELLS 1105 chains, remained unaltered (Figure 5C). Serum levels of soluble CEA increased upon CEA carcinoma installation and decreased to physiological levels upon successful primary tumor elimination (Figure 5D), closely reflecting the human situation. Serum soluble CEA levels, however, increased again after secondary challenge with CEA tumor cells. Titers of anti-cea antibodies of the IgG1 isotype concomitantly increased upon secondary tumor challenge (Figure 5D), indicating the induction of a humoral anti-cea response in addition to a T-cell response. Discussion The study represents first strong evidence in a clinically relevant model that adoptive T-cell therapy targeting CEA is effective toward pancreas carcinoma and can be feasible without inducing severe autoimmune colitis. All mice treated with a single intravenous dose of the SCA431 CAR-engineered T cells showed profound regression of CEA pancreatic cancer; 6 of 8 mice were cured of pancreatic carcinoma in the long term. The anti-tumor response was specifically mediated by the engineered CAR because T cells without CAR or with CAR of irrelevant specificity did not impact tumor progression. The model represents major aspects of clinical relevance, ie, the targeted tumor-associated antigen CEA is a self-antigen and tolerated, is physiologically expressed in various healthy tissues, the CEA pancreatic carcinoma is grown in the pancreas, and soluble CEA is secreted into the serum, all reflecting the clinical situation. T cells engineered with an anti-cea CAR controlled CEA tumor progression in this model. A CAR with a binding domain of moderate avidity, ie, 37 nm, was sufficient to redirect T cells toward CEA pancreatic carcinoma cells. 11 In contrast to the antibody-derived CAR, a recent clinical trial made use of a high-avidity TCR 2 to redirect T cells toward CEA tumor cells. CEA is also expressed by a variety of normal epithelial cells throughout the gastrointestinal and pulmonary tract, preferentially at the luminal side and most prominently by highly differentiated epithelial cells in the upper colonic crypts. Those cells with physiological CEA levels, along with cancer cells with high CEA expression, are also targeted by adoptive cell therapy with redirected T cells. The same observation was made by antibody-redirected therapy. 12,13 Consequently, anti-cea CAR T cells infiltrated the colon and the lung, however, without inducing autoimmune pathology and without attracting an inflammatory infiltrate. Because T cells without CAR or with a CAR of irrelevant specificity did not accumulate in CEA tissues, we conclude that the anti- CEA CAR redirects T cells to the healthy CEA tissues. In contrast to carcinoma, epithelia of healthy tissues express CEA to the luminal site in a strictly polar fashion, making CEA less accessible to infiltrating T cells, and do not result in acute T-cell activation. Accordingly, CAR T cells infiltrating the healthy tissues continued to express the central memory phenotype without further activation, whereas CAR T cells at the pancreatic carcinoma lesion expressed the exhausted CD62L CD127 CD279 phenotype, indicating extensive and prolonged T-cell activation. Lymphodepletion before adoptive T-cell therapy did not induce autoimmune pathology. Although the CEA expression pattern in the CEAtg mouse closely resembles that in humans, the CEA expression levels are 3- to 10- fold higher than physiologically found in the human situation 8 ; another CEAtg strain expresses CEA at 10-fold lower levels. 14 Transfer of CEA-specific T cells into mice of the latter strain likely underestimates the risk of autoimmunity through CEA-specific T-cell therapy, 15 while the model we used can overestimate the situation, as in other studies. 16 Our observation is in contrast to a previous study reporting inflammatory colitis upon adoptive transfer of TCR-redirected T cells into the lymphopenic, irradiated host. 16 Concomitant treatment with anti-cd25 antibodies prevented colitis, while preserving anti-tumor activity. This may be due to dampening the in vivo activation of adoptively transferred T cells when engaging antigen in the CEAtg mouse, lessening the severity of the autoimmune reaction. Reducing the affinity of the targeting domain, as used in our study, and thereby the threshold in T-cell activation, may aim at the same direction. 10 We transferred 10 7 purified CAR-engineered T cells in a single intravenous dose, which was highly effective in eliminating transferred pancreatic carcinoma cells from the pancreas. Although mouse models have their limitations with this respect, the number of transferred T cells per body weight is in the range that has been administered to patients in trials 17 and is 100-fold higher than in a more recent CEA trial. 2 Although we did not observe CAR T-cell mediated damage of healthy CEA tissues in this experimental study, anti-cea CAR T cells heavily accumulated in lung and colon, which is in contrast to the trial in which smaller numbers of engineered T cells produced severe autoimmune pathologies. The same was observed after adoptive transfer of T cells with a high-avidity TCR specific for CEA targeting metastatic colorectal cancer. 2 The TCR was affinity-matured by amino acid substitutions in the TCR -chain. 18 Adoptive therapy with those engineered cells induced objective carcinoma regression in 1 patient; however, induced severe colitis with grades 2 3 diarrhea in all 3 patients, which, although transient, was dose limiting and resulted in trial termination. There are several differences between the clinical trial and our study. First, the trial used a TCR, whereas we used an antibody for redirecting T cells. Different CEA epitopes are targeted; the TCR recognizes the CEA ( ) peptide in the context of HLA-A2.1, the CAR an epitope of the membrane proximal CEA domain independently of the major histocompatibility complex. The CEA peptide is cross-presented by stroma cells lacking CEA expression, and are therefore potential targets for TCR but not for CAR-modified T cells. 19 Second, the TCR was of high avidity, 18 whereas the CAR binding domain was of moderate affinity. Third, colon mucosa of trial patients was

12 1106 CHMIELEWSKI ET AL GASTROENTEROLOGY Vol. 143, No. 4 edematous with areas of ulceration and loss of mucosal folds during cancer progression, thereby making CEA epithelial cells more accessible to engineered T cells. Consequently, T cells infiltrating the mucosa 1 to 2 weeks after therapy onset were fully activated. In the mouse model, the entire colon was of healthy morphology and unaffected by cancer cell infiltrations without signs of disrupted colon mucosa. Although some T-cell infiltrations were clearly detected, no indications of extensive T-cell activation and exhaustion, as found at the pancreatic tumor site, were obvious. Fourth, T cells were transferred into patients after extensive preconditioning. Lymphodepletion before cell therapy seems to be required to sustain persistence of modified T cells in the long term 2,20,21 ; transferred cells did not persist substantially in the clinical situation without preconditioning. Although we did not find autoimmune pathology by CAR T cells after lymphodepleting preconditioning in the experimental model, the reactivity of the adoptively transferred T cells toward healthy epithelia might be more intense under induced lymphopenia due to the lack of repression, eg, by regulatory cells. The impact of lymphodepletion on favoring autoimmune colitis is therefore not clear. In this context, we would like to mention that mice were housed under specifc pathogen free (SPF) conditions, which are far different than the human situation, in which the colon is colonized by commensal bacteria that enhance local immune cell activity. 22 Reduction of the colonic flora before T-cell transfer targeting CEA can be beneficial for avoiding additional immune cell stimulation. T-cell persistence in circulation was reported to correlate with clinical response, 23 whereas other reports do not support the conclusion. 2 In the model used here, bioluminescence imaging revealed T-cell persistence in the affected pancreas more than 3 weeks after adoptive therapy, even when tumor signals were no longer detectable by bioluminescence (cf Figure 3A) and tumor cells could no longer be recorded by histological examinations. We think it more reasonable that the T cells persisted due to a local inflammatory environment. Apart from local persistence, a more general, CEA-specific anti-tumor response persisted for a prolonged period of time, because challenge with CEA, but not with CEA, tumor cells produced secondary tumor rejection in mice, which had received anti-cea CAR T cells and successfully eliminated the primary tumor cell inoculation (cf Figure 5A). The observation is in accordance with a most recent report from our group demonstrating long-term tumor rejection by CD3 CAR T cells, while we transferred CD28- CAR T cells. 24 An antibody response might also be involved in secondary tumor rejection because the serum level of specific anti- CEA IgG1 increased substantially upon secondary tumor challenge (cf Figure 5D). The serum CEA levels increased to about 10 g/l in the CEAtg mouse with CEA pancreatic cancer, which dropped to physiological levels after successful tumor cell elimination (Figure 5D). Increased serum CEA is frequently found in cancer patients and might represent a major issue in the clinical situation because soluble CEA can block T-cell recognition by binding to the anti-cea CAR. The T-cell response toward CEA cancer cells, however, was not blocked by soluble CEA in the CEAtg mouse in vivo and in concentrations up to 25 g/ml in vitro, the latter being in the upper range found in colon and pancreatic cancer patients. This might be due to the fact that the antigen in a membrane-bound form is present in preformed microdomains and thereby more likely generates CAR clustering and formation of a functional synapse than the same antigen in a mono- or dimeric form in solution. We therefore do not expect a major impact of soluble CEA on the CAR redirected T-cell anti-tumor response in the clinical situation. (Supplementary Figure 2). Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at and at dx.doi.org/ /j.gastro References 1. Hariharan D, Saied A, Kocher HM. Analysis of mortality rates for pancreatic cancer across the world. HPB (Oxford) 2008;10: Parkhurst MR, Yang JC, Langan RC, et al. T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 2011; 19: Kochenderfer JN, Wilson WH, Janik JE, et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood 2010;116: Hombach A, Heuser C, Abken H. The recombinant T cell receptor strategy: insights into structure and function of recombinant immunoreceptors on the way towards an optimal receptor design for cellular immunotherapy. Curr Gene Ther 2002;2: Gansauge S, Gansauge F, Beger HG. Molecular oncology in pancreatic cancer. J Mol Med (Berl) 1996;74: Hodge JW. Carcinoembryonic antigen as a target for cancer vaccines. Cancer Immunol Immunother 1996;43: Hombach AA, Schildgen V, Heuser C, et al. T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells. J Immunol 2007;178: Eades-Perner AM, van der PH, Hirth A, et al. Mice transgenic for the human carcinoembryonic antigen gene maintain its spatiotemporal expression pattern. Cancer Res 1994;54: Corbett TH, Roberts BJ, Leopold WR, et al. Induction and chemotherapeutic response of two transplantable ductal adenocarcinomas of the pancreas in C57BL/6 mice. Cancer Res 1984;44: Bruynck A, Seemann G, Bosslet K. Characterisation of a humanised bispecific monoclonal antibody for cancer therapy. Br J Cancer 1993;67: Bosslet K, Steinstraesser A, Schwarz A et al. Quantitative considerations supporting the irrelevance of circulating serum CEA for the immunoscintigraphic visualization of CEA expressing carcinomas. Eur J Nucl Med 1988;14: Hance KW, Zeytin HE, Greiner JW. Mouse models expressing human carcinoembryonic antigen (CEA) as a transgene: evaluation of CEA-based cancer vaccines. Mutat Res 2005;576:

13 October 2012 ADOPTIVE THERAPY WITH CEA-REDIRECTED T CELLS Smith CL, Dulphy N, Salio M, et al. Immunotherapy of colorectal cancer. Br Med Bull 2002;64: Clarke P, Mann J, Simpson JF, et al. Mice transgenic for human carcinoembryonic antigen as a model for immunotherapy. Cancer Res 1998;58: Ojima T, Iwahashi M, Nakamura M, et al. Successful cancer vaccine therapy for carcinoembryonic antigen (CEA)-expressing colon cancer using genetically modified dendritic cells that express CEA and T helper-type 1 cytokines in CEA transgenic mice. Int J Cancer 2007;120: Bos R, van Duikeren S, Morreau H, et al. Balancing between antitumor efficacy and autoimmune pathology in T-cell-mediated targeting of carcinoembryonic antigen. Cancer Res 2008;68: Dudley ME, Yang JC, Sherry R, et al. Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. J Clin Oncol 2008;26: Parkhurst MR, Joo J, Riley JP, et al. Characterization of genetically modified T-cell receptors that recognize the CEA: peptide in the context of HLA-A2.1 on human colorectal cancer cells. Clin Cancer Res 2009;15: Junghans RP. Is it safer CARs that we need, or safer rules of the road? Mol Ther 2010;18: Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 2002;298: Robbins PF, Dudley ME, Wunderlich J, et al. Cutting edge: persistence of transferred lymphocyte clonotypes correlates with cancer regression in patients receiving cell transfer therapy. J Immunol 2004;173: Paulos CM, Kaiser A, Wrzesinski C, et al. Toll-like receptors in tumor immunotherapy. Clin Cancer Res 2007;13: Kalos M, Levine BL, Porter DL, et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 2011;3:95ra Chmielewski M, Rappl G, Hombach AA, Abken H. T cells redirected by a CD3 chimeric antigen receptor can establish self-antigen-specific tumour protection in the long term. Gene Ther 2012 [Epub ahead of print]. Received September 27, Accepted June 21, Reprint requests Address requests for reprints to: Prof Hinrich Abken, University of Cologne, Center for Molecular Medicine Cologne, Robert-Koch-Str. 21, D Cologne, Germany. hinrich.abken@uk-koeln.de; fax: 49 (0) Acknowledgments We thank Dr David Gilham and the Central Animal Facility, Patterson Institute for Cancer Research, Manchester, UK, for providing us with CEAtg mice. Conflict of interest The authors disclose no conflicts. Funding Work was funded by the Deutsche Krebshilfe, Bonn, Germany, the Wilhelm Sander-Stiftung, München, and the Else Kröner-Fresenius Stiftung, Homburg v.d.h., Germany.

14 1107.e1 CHMIELEWSKI ET AL GASTROENTEROLOGY Vol. 143, No. 4 Supplementary Figure 1. No substantial change in body weight of mice after adoptive therapy with CAR T cells. Pancreas carcinomas were induced by intra-pancreatic injection of the CEA or the CEA Panc02 carcinoma cells as described in Figure 3. When tumors were established, anti-cea CAR T cells were injected into the tail vein at day 0 (10 7 cells/mouse). For comparison, T cells without CAR were injected. Up to eight mice were treated in each cohort. The body weight of each mouse was recorded during treatment. Data represent the mean.

15 October 2012 ADOPTIVE THERAPY WITH CEA-REDIRECTED T CELLS 1107.e2 Supplementary Figure 2. No inflammatory response at the targeted tumor site after adoptive therapy with CAR T cells. Pancreatic tumors were induced and T cells with or without anti-cea CAR were adoptively transferred as described in Figure 3. Tissue sections of the pancreatic tumor site as well as of the large and small intestine and the lung were recorded by staining for MPO, IL-1, and TNF-.